Article
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*e-mail: pablo.machado@pucrs.br; diogenes@pucrs.br #These authors contributed equally to this work
Synthesis and Evaluation of Thiazolyl-1
H
-benzo[
d
]imidazole Inhibitors of
Mycobacterium tuberculosis
Inosine Monophosphate Dehydrogenase
Kenia Pissinate,a,# Diana Carolina Rostirolla,a,b,# Laura Miranda Pinheiro,a
Priyanka Suryadevara,c Perumal Yogeeswari,c Dharmarajan Sriram,c
Luiz Augusto Basso,a,b Pablo Machado*,a,d and Diógenes Santiago Santos*,a,b
aCentro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul,
90619-900 Porto Alegre-RS, Brazil
bPrograma de Pós-Graduação em Medicina e Ciências da Saúde, Pontifícia Universidade Católica do Rio Grande do Sul, 90619-900 Porto Alegre-RS, Brazil
cDrug Discovery Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, 500078 Hyderabad, India
dPrograma de Pós-Graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul, 90619-900 Porto Alegre-RS, Brazil
Using an orthologue-based design approach, we synthesized and assayed a series of thiazolyl-1H-benzo[d]imidazole derivatives as inhibitors of Mycobacterium tuberculosis inosine 5’-monophosphate dehydrogenase (MtIMPDH). From these experiments, a benzo[d] imidazole compound was described to inhibit the enzyme in the low micromolar range
(Ki IMP = 0.55 ± 0.02 µM), which places this compound among the most potent in vitro MtIMPDH
inhibitors developed to date. In addition, steady-state kinetic measurements and docking simulations were employed to determine its inhibition and interaction modes. The results described herein may be useful for the design and development of novel alternative therapeutics for tuberculosis that target MtIMPDH, a predicted to be essential (for optimal in vitro bacillus growth), druggable and assayable molecular target.
Key words: drug research, tuberculosis, orthologue-based design, IMPDH inhibitors
Introduction
“Tuberculosis (TB) remains a major global health problem”.This affirmation opens the last TB report of the World Health Organization (WHO).1 Declared since the 1990s to be a global public health emergency, human TB has stricken an estimated 9.0 million people, which resulted in 1.5 million deaths worldwide in 2013.1 Moreover, it has been estimated that approximately one third of the world’s population is currently infected with a latent or dormant form of Mycobacterium tuberculosis (Mtb), the main causative agent of TB in humans, thus increasing the number of people at risk for developing active TB due to infection reactivation.2 The emergence of multi
and extensively drug-resistant strains of Mtb (MDR-TB and XDR-TB, respectively),3 the variable efficacy of the current vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG),4 and the increasing prevalence of TB-HIV co-infection1 have highlighted the need for new control measures. Although innovative drugs such as bedaquiline5,6 and repurposing old drug classes have been described as a promising strategy to address this problem,7 the current therapeutic TB treatments are suboptimal and new drugs are still needed.8
study of novel targets can conduce to new molecules with novel mechanisms of action that are active against resistant and latent forms of Mtb, both of which are crucial attrition points in TB treatment.
Supported by the essentiality of most of its biosynthetic steps for optimal in vitro Mtb growth11 and by evidence of nucleotide synthesis maintenance in latent bacillus,12 the gene products of the purine nucleotide biosynthesis pathway have emerged as potential targets for drug development against both active and latent TB.13 Among the enzymes that compose this pathway, the inosine 5’-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes the penultimate and rate-limiting step in guanine nucleotide biosynthesis: the oxidation of inosine 5’-monophosphate (IMP) to xanthosine 5’-monophosphate (XMP) and the concomitant reduction of nicotinamide adenine dinucleotide (NAD+) (Scheme 1).
Encoded by the guaB2 gene (Rv3411c),14 Mycobacterium
tuberculosis IMPDH (MtIMPDH) has been predicted to be essential for optimal in vitro growth of M. tuberculosis
H37Rv.11,15 Additionally, prokaryotic and eukaryotic IMPDHs have distinct structural features and kinetic properties, which make the development of species-selective inhibitors feasible.16 The different inhibition patterns of
MtIMPDH and human IMPDH type II (HsIMPDH-II)17 have supported the proposition of this target for the development of alternative drugs with selective toxicity, a basic pharmacology principle. Moreover, both proteins share sequence similarity of approximately 38%, and the amino acids residues at the HsIMPDH-II nicotinamide sub site that account for sensitivity to potent drugs, such as mycophenolic acid, are not conserved in the MtIMPDH.18
Recently, the potential of IMPDH inhibitors in antimicrobial chemotherapy has been exploited, highlighting the applicability of this enzyme in drug discovery campaigns.19-21 These findings have described active compounds in whole cell assays that generate lead molecules that are now candidates for further development.14,19
Thiazolyl-1H-benzo[d]imidazole derivatives have been identified from high-throughput screens as
selective inhibitors of Cryptosporidium parvum IMPDH (CpIMPDH) that target the diverged NAD+ site of enzyme.22 Diffractometry studies from the crystal structure of
CpIMPDH (PDB code: 3KHJ) in complex with IMP and a benzo[d]imidazole-based compound (named C64) have highlighted amino acid interactions, which account for the selectivity of these compounds to CpIMPDH over human IMPDH.23 Amino acid sequence alignment of MtIMPDH and CpIMPDH showed that almost all of the identified residues that interact with C64 (except Ser354, which was replaced by Ala483 in MtIMPDH) are conserved between the sequences (Figure S1 of the Supplementary Information). Incidentally, MtIMPDH has been predicted to be sensitive to CpIMPDH inhibitors16 because phylogenetic analysis points to lateral gene transfer from bacteria, thus making eukaryotic IMPDH closely related to their prokaryotic counterparts.24
Therefore, there is an expected divergence between mycobacteria and host IMPDH enzymes that, taken together with the sequence conservation of binding sites, prompted us to investigate the interaction of benzo[d]imidazole derivatives with MtIMPDH. For this, we deemed it appropriate to synthesize a series of thiazolyl-1H-benzo[d]imidazoles and evaluate their in vitro potential as inhibitors of MtIMPDH in an orthologue-based design approach.
Results and Discussion
The synthesis of thiazolyl-1H-benzo[d]imidazoles was carried out in two reaction steps in accordance to previously described protocol (Scheme 2).25 It is noteworthy that proposed structural modifications were aimed primarily at the creation of an electron density gradient, variation of the molecular volume, and to explore the importance of the aromaticity and planarity of the substituents in a preliminary SAR (structure activity relationship) study.
First, the classical substitution reaction of primary and secondary amines 1 at the carbonyl group of bromoacetyl chloride 2 in the presence of catalytic
NH
N N
O
N
O
OH H H
H H
OH O P
-O
O
O
-NH
N H N
O
N
O
OH H H
H H
OH O P
-O
O
O
-O
H2O IMPDH NADH
NAD+ 2H+
IMP XMP
amounts of 4-dimethyl aminopyridine (DMAP) conduced to 2-bromo-N-phenylacetamides 3a-l with 50-85% yield. The aliphatic nucleophilic substitution (SN2) of
2-bromo-N-phenylacetamides with 2-(4-thiazolyl)benzimidazole (4) using potassium carbonate (K2CO3) as base and dimethyl formamide (DMF) as solvent furnished the desired thiazolyl-1H-benzo[d]imidazoles 5a-l with 15-88% yield. All spectroscopic and spectrometric data obtained were in agreement with the proposed structures (see Supplementary Information).
The effect of each compound on catalytic activity of recombinant MtIMPDH was evaluated by monitoring the production of NADH (nicotinamide adenine dinucleotide). First, all of the synthesized compounds were assayed at a final-fixed concentration of 10 µM, and when the inhibition was greater than 50% of the initial enzymatic activity, the IC50 values (concentration of inhibitor that reduces enzyme velocity by half) were determined (Table 1).
The results shown in the Table 1 demonstrate that
MtIMPDH inhibition is highly sensitive to minor modifications of the thiazolyl-1H-benzo[d]imidazoles substituent groups. Additionally, the electron withdrawing groups generally showed better inhibitory activity on recombinant MtIMPDH. Compared with the non-substituted
5a, the replacement of hydrogen by halogens at the 4-position of the benzene ring furnished molecules with
improved inhibitory activity. This strategy has been extensively explored in medicinal chemistry programs aiming for more lipophilic molecules and the addition of a new site for hydrogen and/or halogen bonding. It is noteworthy that as the atomic volume of halogens increased, the inhibitory action of the compounds became
Compound 1,3,5 R1 R2
a Ph H
b 4-F-C6H4 H
c 4-Cl-C6H4 H
d 3-Cl-C6H4 H
e 4-Br-C6H4 H
f 4-I-C6H4 H
g 4-O2N-C6H4 H
h 4-NC-C6H4 H
i 4,2-(MeO)2-C6H3 H
j 3-PrO-C6H4 H
k 2-Me-C6H4 H
l CH2(CH2)3CH2
Scheme 2. Reaction conditions: (i) DMAP, CH2Cl2, 0-25 °C, 3.5 h; (ii) K2CO3, DMF, 25 °C, 8 h.
N N
N S
O
N
R1 R1
HN
1a l- 3a l
-5a l -Cl
O
Br
2
N O
Br
R1 2
R2
HN N
N S
4
50-85%
R2 i
15-88% ii
Table 1.Inhibition of thiazolyl-1H-benzo[d]imidazoles 5a-l on recombinant MtIMPDH activity
entry Inhibition / %a IC
50 / (µmol L–1)
5a < 2% > 10
5b 25% > 10
5c 67% 4.5 ± 0.4b
5d < 2% > 10
5e 80% 3.0 ± 0.3b
5f 60% 9.0 ± 3.0
5g 64% 8.0 ± 0.3
5h 50% ≥ 10
5i < 4% > 10
5j 65% 5.1 ± 0.5
5k < 4% > 10
5l < 3% > 10
aObserved at a final concentration of 10 µmol L–1;bIC
more potent (Table 1). The compound 4-fluoro substituted
5b inhibited the enzyme activity with an IC50 > 10 µmol L–1 (25% at 10 µmol L–1), whereas both 4-chloro (5c) and 4-bromo (5e) substituted compounds conduced to IC50 values of 4.5 ± 0.4 µmol L–1 and 3.0 ± 0.3 µmol L–1, respectively. Interestingly, the iodine substituted compound
5f showed less inhibitory activity on MtIMPDH than did its isostere molecules 5c and 5e; its IC50 was determined at 9.0 ± 3.0 µmol L–1. The lower activity may be related to a possible steric hindrance at the 4-position of the benzene ring of thiazolyl-1H-benzo[d]imidazoles because a molecular volume-improving limit may have been achieved with the bromo-substituted 5e. It is important to note that the replacement of the chloro group attached at the 4- to 3-position of the benzene ring led to compound
5d, which lacked inhibition capacity on MtIMPDH in our experimental conditions. This result emphasizes the fine tuning of the halogen’s position relative to the MtIMPDH binding site. The 4-nitro and 4-cyanobenzo[d]imidazole compounds (5g, 5h) both showed similar inhibition activity, with an IC50 of 8.0 ± 0.3 µmol L–1 and ≥ 10 µmol L–1, respectively. Focusing on the compounds substituted with electron donating groups, the results for benzo[d] imidazoles 5i-k showed that only the compound carrying the 3-propoxy group (5j) exhibited a significant inhibition of MtIMPDH activity. The IC50 of 5j was 5.1 ± 0.5 µmol L–1, similar to lead compound 5e, suggesting the existence of a hydrophobic portion accessed by the mobile 3-propoxy group. Finally, the benzo[d]imidazole derivative from piperidine (5l) did not show action on catalytic MtIMPDH activity in our assay conditions. Therefore, the initial screening of 12 molecules showed that five compounds (5c, 5e-g, and 5j) displayed reasonably good inhibition (IC50 < 10 µmol L–1) on MtIMPDH activity. These five
benzo[d]imidazoles showed no time-dependent inhibition up to 30 min of preincubation with MtIMPDH suggesting rapid equilibrium processes (data not shown). It is noteworthy that compounds 5c and 5e showed selective
MtIMPDH inhibition because both have been described to show an IC50 ≥ 20 µmol L–1for HsIMPDH-II.23
Accordingly, Ki (inhibition constant) measurements were carried out using classic Michaelis-Menten experiments. The Ki values were determined for benzo[d]imidazole 5e, the lead of the series of synthesized analogs. Lineweaver-Burk plots suggested the mode of inhibition based on the effects on Vmax (maximal velocity) and Km (Michaelis constant) values for each inhibition type, and data fitting to appropriate equations led to values for the inhibition constants (Kis and/or Kii; Kii is the overall inhibition constant for the enzyme-substrate-inhibitor complex, and Kis is the overall inhibition constant for the enzyme-inhibitor complex). Particular signatures and mechanism of inhibition for MtIMPDH inhibitors were investigated and provided insights into the interaction mode for the enzyme.14, 17, 19
For benzo[d]imidazole 5e, the types of inhibition were characterized as uncompetitive toward IMP and noncompetitive to NAD+ (Figure 1). Compound 5e showed Kis values of 0.55 ± 0.02 µmol L–1 for IMP and values of Kis = 2 ± 1 µmol L–1 and Kii = 0.7 ± 0.1 µmol L–1 for NAD+. It is important to note that the inhibition profile exhibited by 5e cannot be overcome by improving the substrate concentrations, a detrimental condition for the success of inhibitors aiming for in vivo activity.
A steady-state ordered BiBi kinetic mechanism has been suggested for MtIMPDH, in which IMP binds first and is followed by the binding of NAD+ and NADH dissociates first and is followed by XMP release.26 After the NAD+ binding event, a rapid electron transference forming NADH
and the covalent intermediate E-XMP* for IMPDHs has been reported.27
Similar to the analogous compounds already reported to be CpIMPDH inhibitors, the inhibition pattern showed by 5e indicates that this benzo[d]imidazole binds to both E·IMP and E-XMP*, with a slight preference for the E-XMP* state (Kis > Kii).22 It is interesting to note that the experimental findings concerning the benzo[d]imidazole potencies suggest a similar interaction mode of compounds with MtIMPDH to that shown by C64 with CpIMPDH.23
Finally, 5e inhibits the MtIMPDH with 50-fold less potency than CpIMPDH (IC50 = 0.06 ± 0.03 µmol L–1).25 This difference may be related to the replacement of Ser354 residue in the eukaryotic protein by Ala483 in the
MtIMPDH. This modification alters the physicochemical properties of the binding site because alanine is a non-polar residue replacing a polar one. Thus, the hydrogen-bonding network formed among Glu329, Ser354, Thr221 and amide group23 would not likely occur in MtIMPDH.
In order to explore the binding phenomenon, a three dimensional protein model was constructed and used for docking simulations. The necessary coordinates for the protein structure was prepared using popular method of homology modeling, details of which has been provided in the Supplementary Information. Interestingly, compound 5e
(most active out of 12) was found to be binding to the protein with best ranking and docking score of −6.69 kcal moL-1. The compound was found to be involved in hydrogen bonding with residues Ser83, Ser339, Ile340 and Tyr421 and also in cation-π interaction between Arg443 and phenyl ring of compound (Figure 2). Moreover, the 4-bromo phenyl group of compound was found to be stabilized by strong hydrophobic interactions with residues Met85, Cys341, Met424 and Arg443. These strong interactions of 5e at the active site can be reasoned for its activity towards MtIMPDH. While analyzing the docking pattern of inactive compounds such as 5a and 5d (Figure S7 of the Supplementary Information), the compounds were found to be lacking some of the polar contacts with Ile340 and Tyr421 and also Ser339. The compounds were also found to be orienting slightly in different manner, compared to 5e, which moved the thiazole ring away from Tyr341 making it unavailable for hydrogen bonding.
Conclusions
In summary, we have reported the efficient preparation of benzo[d]imidazole derivatives and their use as inhibitors on catalytic activity of MtIMPDH. The proposition of these compounds as possible MtIMPDH inhibitors was based on their previously described action on CpIMPDH conducing
to selective inhibitors at a low micromolar range. To the best of our knowledge, the titled compounds are among the most potent MtIMPDH inhibitors described to date. Therefore, the data herein presented may provide key insights into the proposition of new compounds that can be used in the further development of novel TB drugs that target MtIMPDH.
Experimental
All common reactants and solvents were used as obtained from commercial suppliers without further purification. All reactions involving reactants, reagents or intermediates sensitive to air or moisture were performed under an inert atmosphere of argon. Melting points were determined using a Microquímica MQAPF-302 apparatus. 1H NMR spectra were acquired on an Anasazi EFT-60 spectrometer (1H at 60.13 MHz) and at 30 °C. 13C NMR spectra were acquired on a Fourier 300 spectrometer of University of Caxias do Sul (UCS) in Brazil (13C at 75.48 MHz) and at 25 °C. DMSO-d6 was used as the solvent, and TMS was used as an internal standard in 5 mm samples tubes. Chemical shifts are expressed in ppm, and
J values are given in Hz. High-resolution mass spectra (HRMS) were obtained for all compounds on an LTQ Orbitrap Discovery mass spectrometer from Thermo Fisher Scientific. This hybrid system combines the LTQ XL linear ion trap mass spectrometer with an Orbitrap mass analyzer. The experiments were performed using direct infusion of
the sample in a solution of acetonitrile (50%), methanol (50%), and formic acid (0.1%), with a flow of 5 µL min-1 in positive-ion mode using electrospray ionization. Elemental composition calculations were performed using a specific tool included in the Qual Browser module of the Xcalibur (Thermo Fisher Scientific, release 2.0.7) software. Fourier transform infrared (FTIR) spectra were recorded using a universal attenuated total reflectance (UATR) attachment on a PerkinElmer Spectrum 100 spectrometer in the wavenumber range of 650-4000 cm-1 with a resolution of 4 cm-1. Purity of compounds was determined by HPLC using an Äkta HPLC system of GE Healthcare® Life Sciences. HPLC analysis conditions: RP column 5 µm Nucleodur C-18 (250 × 4.6 mm); flow rate 1.5 mL min-1; UV detection at 254 nm; 100% water (0.1% acetic acid) for 7 minutes followed by linear gradient from 100% water (0.1% acetic acid) to 90% acetonitrile/methanol (1:1, v/v) in 16 min; the last partition was maintained by 15 min and subsequently returned to 100% water (0.1% acetic acid) in 5 min remaining for additional 6 min. All evaluated compounds were > 95% pure.
General procedure for the preparation of
2-bromo-N-phenylacetamides 3a-l
To a solution containing aniline 1 (0.382 g, 4.1 mmol) and a catalytic amount of dimethylaminopyridine (DMAP) (0.150 g, 30 mmol%) in dry dichloromethane (20 mL) at 0 °C was added bromoacetyl chloride 2 (0.425 mL, 5.1 mmol) dropwise. The resulting solution was stirred at 0 °C for 30 min, and the temperature was then increased to 25 °C. After stirring for an additional 3 h, the reaction mixture was diluted with diethyl ether (50 mL). All of the stirring time was accomplished under argon atmosphere. The organic layers were washed sequentially with a solution of HCl (1 mol L-1, 2 × 50 mL), water (1 × 100 mL), saturated aqueous NaHCO3 (3 × 50 mL), and brine (5% m/v, 1 × 50 mL). Finally, the organic solution was dried over anhydrous MgSO4 and evaporated under vacuum, and the residue was purified by flash chromatography on silica gel eluting with chloroform:methanol (40:1).
General procedure for the preparation of
2-thiazol-4-yl-1H-benzo[d]imidazoles 5a-l
Selected example for compound N
-phenyl-2-(2-thiazol-4-yl)-1H-benzo[d]imidazole-1-yl)acetamide (5a).
A solution containing the 2-bromo-N-phenyl-acetamide
3a (0.107 g, 0.5 mmol), of K2CO3 (0.540 g, 1.56 mmol), and 2-(4-thiazolyl)benzimidazole 4 (0.103 g, 0.51 mmol) in 4 mL of dimethylformamide (DMF) was stirred for 8 h at
25 °C. After, the reaction mixture was dissolved in 200 mL of distilled water. The precipitated product was filtered off, washed with water and dried under vacuum. Purification of the compound was accomplished by flash chromatography on silica gel (Macherey-Nagel, 35-70 mesh) eluting with chloroform:methanol (40:1). 2-Thiazol-4-yl-1H-benzo[d] imidazoles 5b-l were prepared in accordance with the above described protocol.
N-Phenyl-2-(2-(thiazol-4-yl)-1H-benzo[d]imidazole-1-yl)
acetamide (5a)
Yield 35%; mp 219-220 °C; IR (UATR) ν / cm-1 1670 (C=O); 1H NMR (60 MHz, DMSO-d
6) d 5.72 (s, 2H, CH2), 7.00-7.64 (m, 9H, Bi-H*, Ph-H), 8.57 (d, 1H, J 2.1, Th-H*), 9.30 (d, 1H, J 2.1 Hz, Th-H), 10.53 (br, 1H, NH); HRMS (FTMS + pESI) m/z calcd. for C18H14N4OS (M)+: 335.0961; found: 335.0946; Bi-H*: benzoimidazole hydrogens; Th-H*: thiazole hydrogens.
N-(4-Fluorphenyl)-2-(2-(thiazol-4-yl)-1H-benzo[d]imidazol-1-yl)
acetamide (5b)
Yield 75%; mp 227- 228 °C; IR (UATR) ν / cm-1 1673 (C=O); 1H NMR (60 MHz, DMSO-d
6) d 5.68 (s, 2H, CH2), 6.96-7.72 (m, 8H, Bi-H*, C
6H4), 8.57 (d, 1H, J 2.1 Hz, Th-H*), 9.30 (d, 1H, J 2.1 Hz, Th-H); HRMS (FTMS + pESI)
m/z calcd. for C18H13FN4OS (M)+: 353.0867; found: 353.0847; Bi-H*: benzoimidazole hydrogens; Th-H*: thiazole hydrogens.
N-(4-Chlorophenyl)-2-(2-(thiazol-4-yl)-1H-benzo[d
]imidazol-1-yl)acetamide (5c)
Yield 85%; mp 228-229 °C; IR (UATR) ν / cm-1 1682 (C=O); 1H NMR (60 MHz, DMSO-d
6) d 5.71 (s, 2H, CH2), 7.19-7.80 (m, 8H, Bi-H*, C
6H4), 8.57 (d, 1H, J 2.1 Hz, Th-H*), 9.03 (d, 1H, J 2.1 Hz, Th-H); 13C NMR (75 MHz, DMSO-d6) d 48.6 (CH2), 111.1, 119.4, 121.1, 122.7, 122.8, 123.3, 127.4, 129.2, 136.9, 138.2, 142.7, 147.3, 147.5, 155.8, 166.5 (C=O); HRMS (FTMS + pESI) m/z calcd. for C18H13ClN4OS (M)+: 369.0563; found: 369.0555; Bi-H*: benzoimidazole hydrogens; Th-H*: thiazole hydrogens.
N-(3-Chlorophenyl)-2-(2-(thiazol-4-yl)-1H-benzo[d
]imidazol-1-yl)acetamide (5d)
Yield 18%; mp 211 °C; IR (UATR) ν / cm-1 1696 (C=O); 1H NMR (60 MHz, DMSO-d
6) d 5.72 (s, 2H, CH2), 7.20-7.78 (m, 8H, Bi-H*, C
6H4), 8.59 (d, 1H, J 2.1 Hz, Th-H*), 9.31 (d, 1H, J 2.1 Hz, Th-H), 10.64 (br, 1H, NH); 13C NMR (75 MHz, DMSO-d
(M)+: 369.0571; found: 369.0563; Bi-H*: benzoimidazole hydrogens; Th-H*: thiazole hydrogens.
N-(4-Bromophenyl)-2-(2-(thiazol-4-yl)-1H-benzo[d
]imidazol-1-yl)acetamide (5e)
Yield 70%; mp 231 °C; IR (UATR) ν / cm-1 1682 (C=O); 1H NMR (60 MHz, DMSO-d
6) d 5.70 (s, 2H, CH2), 7.19-7.79 (m, 8H, Bi-H*, C
6H4), 8.57 (d, 1H, J 2.1 Hz, Th-H*), 9.30 (d, 1H, J 2.1 Hz, Th-H); HRMS (FTMS + pESI) m/z calcd. for C18H13BrN4OS (M)+: 413.0066; found: 413.0045; Bi-H*: benzoimidazole hydrogens; Th-H*: thiazole hydrogens.
N-(4-Iodophenyl)-2-(2-(thiazol-4-yl)-1H-benzo[d
]imidazol-1-yl)acetamide (5f)
Yield 43%; mp 258 °C; IR (UATR) ν / cm-1 1675 (C=O); 1H NMR (60 MHz, DMSO-d
6) d 5.89 (s, 2H, CH2), 7.18-7.74 (m, 8H, Bi-H*, C
6H4), 8.54 (d, 1H, J 2.1 Hz, Th-H*), 9.27 (d, 1H, J 2.1 Hz, Th-H); 13C NMR (75 MHz, DMSO-d6) d 48.0 (CH2), 86.9, 110.7, 118.9, 121.2, 122.2, 122.3, 122.8, 136.5, 137.5, 138.6, 142.2, 146.8, 147.0, 155.3, 166.1 (C=O); HRMS (FTMS + pESI) m/z calcd. for C18H13IN4OS (M)+: 460.9928; found: 460.9938; Bi-H*: benzoimidazole hydrogens; Th-H*: thiazole hydrogens.
N-(4-Nitrophenyl)-2-(2-(thiazol-4-yl)-1H-benzo[d
]imidazol-1-yl)acetamide (5g)
Yield 88%; mp 167 °C; IR (UATR) ν / cm-1 1696 (C=O); 1H NMR (60 MHz, DMSO-d
6) d 5.77 (s, 2H, CH2), 7.20-7.36 (m, 2H, Bi-H*), 7.61-7.87 (m, 4H, Bi-H, C
6H4) 8.14-8.30 (d, 2H, J 9.6 Hz, C6H4), 8.59 (d, 1H, J 2.1 Hz, Th-H*), 9.22 (d, 1H, J 2.1 Hz, Th-H); 13C NMR (75 MHz, DMSO-d6) d 48.7 (CH2), 110.7, 118.8, 122.1, 122.3, 122.8, 125.0, 136.4, 142.0, 142.2, 146.7, 147.0, 155.3, 167.2 (C=O); HRMS (FTMS + pESI) m/z calcd. for C18H13N5O3S (M)+: 380.0812; found: 380.0805; Bi-H*: benzoimidazole hydrogens; Th-H*: thiazole hydrogens.
N-(4-Cyanophenyl)-2-(2-(thiazol-4-yl)-1H-benzo[d
]imidazol-1-yl)acetamide (5h)
Yield 66%; mp 236 °C; IR (UATR) ν / cm-1 1709 (C=O); 1H NMR (60 MHz, DMSO-d
6) d 5.70 (s, 2H, CH2), 7.18-7.74 (m, 8H, Bi-H*, C6H4), 8.57 (d, 1H,
J 2.1 Hz, Th-H*), 9.29 (d, 1H, J 2.1 Hz, Th-H); HRMS (FTMS + pESI) m/z calcd. for C19H13N5OS (M)+: 360.0914; found: 360.0895; Bi-H*: benzoimidazole hydrogens; Th-H*: thiazole hydrogens.
N-(2,4-Dimethoxyphenyl)-2-(2-(thiazol-4-yl)-1H-benzo[d]
imidazol-1-yl)acetamide (5i)
Yield 27%; mp 234 °C; IR (UATR) ν / cm-1 1671 (C=O); 1H NMR (60 MHz, DMSO-d
6) d 3.74 (s, 3H, OCH3), 3.86
(s, 3H, OCH3), 5.72 (s, 2H, CH2), 6.35-6.62 (m, 2H, C6H3), 7.21-7.76 (m, 5H, Bi-H*, C
6H3), 8.57 (d, 1H, J 2.1 Hz, Th-H*), 9.35 (d, 1H, J 2.1 Hz, Th-H); 13C NMR (75 MHz, DMSO-d6) d 48.1 (CH2), 55.3, 55.8, 98.8, 103.9, 110.6, 118.9, 120.1, 122.3, 122.4, 122.7, 122.8, 136.3, 142.3, 146.9, 147.0, 151.0, 155.3, 156.7, 165.6 (C=O); HRMS (FTMS + pESI) m/z calcd. for C20H18N4O3S (M)+: 395.1172; found: 395.1177; Bi-H*: benzoimidazole hydrogens; Th-H*: thiazole hydrogens.
N-(3-Propoxyphenyl)-2-(2-(thiazol-4-yl)-1H-benzo[d]
imidazol-1-yl)acetamide (5j)
Yield 15%; mp 223 °C; IR (UATR) ν / cm-1 1686 (C=O); 1H NMR (60 MHz, DMSO-d
6) d 0.93 (t, 3H, J 7 Hz, CH3), 1.65 (quint, 2H, J 7 Hz, CH2), 3.84 (t, 2H, J 7, CH2), 5.75 (s, 2H, CH2), 6.53-7.67 (m, 8H, Bi-H*, C6H4), 8.56 (d, 1H,
J 2.1 Hz, Th-H*), 9.30 (d, 1H, J 2.1 Hz, Th-H). 10.42 (br, 1H, NH); 13C NMR (75 MHz, DMSO-d
6) d 10.4, 22.0, 48.0 (CH2), 68.8, 105.2, 109.5, 111.1, 122.3, 122.8, 129.6, 140.0, 159.0, 165.9 (C=O); HRMS (FTMS + pESI) m/z calcd. for C21H20N4O2S (M)+: 393.1380; found: 393.1378; Bi-H*: benzoimidazole hydrogens; Th-H*: thiazole hydrogens.
2-(2-(Thiazol-4-yl)-1H-benzo[d]imidazol-1-yl)-N
-o-tolylacetamide (5k)
Yield 40%; mp 165 °C; IR (UATR) ν / cm-1 1666 (C=O); 1H NMR (60 MHz, DMSO-d
6) d 2.21 (s, 3H, CH3), 5.70 (s, 2H, CH2), 6.91-7.77 (m, 8H, Bi-H*, C6H4), 8.53 (d, 1H,
J 2.1 Hz, Th-H*), 9.30 (d, 1H, J 2.1 Hz, Th-H), 9.71 (br, 1H, NH); HRMS (FTMS + pESI) m/z calcd. for C19H16N4OS (M)+: 349.1118; found: 349.1122; Bi-H*: benzoimidazole hydrogens; Th-H*: thiazole hydrogens.
1-(Piperidin-1-yl)-2-(2-(thiazol-4-yl)-1H-benzo[d
]imidazol-1-yl)ethanone (5l)
Yield 44%; mp 171 °C; IR (UATR) ν / cm-1 1642 (C=O); 1H NMR (60 MHz, DMSO-d
6) d 1.63 (br, 6H, Pi-H*), 3.51 (br, 6H, Pi-H), 5.64 (s, 2H, CH2), 7.25-7.81 (m, 4H, Bi-H*), 8.31 (d, 1H, J 2.1 Hz, Th-H*), 8.83 (d, 1H, J 2.1 Hz, Th-H); 13C NMR (75 MHz, DMSO-d
6) d 24.1, 25.3, 25.9, 42.7, 45.4, 46.4, 110.6, 118.7, 122.0, 122.2, 122.6, 136.5, 142.0, 146.9, 147.1, 155.0, 164.9 (C=O); HRMS (FTMS + pESI) m/z calcd. for C17H18N4OS (M)+: 327.1274; found: 327.1273; Pi-H: piperidine hydrogens; Bi-H*: benzoimidazole hydrogens; Th-H*: thiazole hydrogens.
Enzymatic activity assays
a UV-2550 UV-Visible spectrophotometer (Shimadzu) by monitoring NAD+ reduction to NADH at 340 nm (εβ−NADH: 6.22 M-1 cm-1) and corrected for non-catalyzed chemical reactions in the absence of MtIMPDH. Assays were performed at 37 °C, in 50 mmol L–1 Tris pH 8.5, 1 mmol L–1 DTT, and 200 mmol L–1 KCl. Enzyme-catalyzed reaction started with the addition of
MtIMPDH at 145 nM to the assay mixture (500 µL of final volume) and data were collected for 5 min. The inhibitors were dissolved in dimethyl sulfoxide (DMSO) and added to the reaction mixture at a final concentration of 10 µmol L–1. Enzyme velocity was used to determine the % inhibition and, as a control, the maximal rate of the enzymatic reaction (100% of MtIMPDH activity) was determined in the absence of inhibitor and in the presence of fixed non-saturating concentrations of IMP (100 µmol L–1, K
0.5 ca. 120 µmol L–1) and NAD+ (1 mmol L–1, K
m ca.887 µmol L–1), in the presence of 1% DMSO.26 Compounds able to reduce the enzyme activity by more than 50% were selected for further IC50 determination.
IC50 determinations
The IC50 values were determined by adding different concentrations of the compounds (dissolved in DMSO) to the reaction mixture above described and the enzyme velocity was used to determine the % inhibition. The IC50 values were estimated using equation 1, where [I] is the inhibitor concentration, vi is the initial velocity in the presence of the inhibitor, vo is the initial velocity in the absence of inhibitor, and the IC50 value is defined as the concentration of the inhibitor that reduces the enzyme velocity by half.
+ = 50
0 [ ]
1 1 IC I v vi (1) Time-dependent inhibition
To evaluate whether or not enzyme inhibition may be time dependent, recombinant MtIMPDH was preincubated at room temperature with 10 µmol L–1 inhibitor (final concentration), which was then added at different times (up to 30 min) to the reaction mixture, as previously described. This analysis was performed to determine if inhibition follows a rapid equilibrium mode (classical competitive, uncompetitive and noncompetitive inhibition) or if there is a slow step in the equilibrium process.
Mode of inhibition and determination of the overall inhibition
constant for 5e
The determination of Ki values and the mode of inhibition were performed for 5e inhibitor towards both MtIMPDH substrates. Lineweaver-Burk (double-reciprocal) plots were employed to determine the mode of inhibition (competitive, noncompetitive or uncompetitive) and data fitting to appropriate equations gave values for the inhibition constants (Kis and/or Kii). In short, the inhibition mode was proposed based on the effects on Vmax and Km values for each inhibition type, resulting in plots with distinct straight line patterns toward either IMP or NAD+ as follows: lines intercept on y axis for competitive inhibition (does not affect the apparent Vmax and increases apparent Km), lines intercept on left of y axis for noncompetitive inhibition (decreases apparent Vmax, and does not affect apparent Km values if Kis = Kii, increases the apparent Km values if Kis < Kii, or decreases the Km values if Kis > Kii), and parallel lines for uncompetitive inhibition (decreases both apparent Vmax and Km values).28
The inhibition studies were carried out at varying concentrations of one substrate, a fixed non-saturating concentration of the other substrate, and in the absence of inhibitor and in the presence of three different concentrations of each inhibitor 5e (0.5, 1 and 1.5 µmol L–1). For IMP substrate analysis, the experiment was carried out with IMP concentrations ranging from 40 µmol L–1 to 1000 µmol L–1 and NAD+ at 1 mmol L–1; for NAD+ substrate analysis, the NAD+ concentrations ranged from 0.2 mmol L–1 to 4 mmol L–1, with IMP fixed at 100 µmol L–1. The enzyme concentration was constant at 550 nM throughout the assays. Noncompetitive and uncompetitive inhibition data were fitted to equations 2 and 3, respectively:
+ + + = ii is m K I V S K I V K v ] [ 1 1 ] [ 1 1 1 max max 0 (2) + + = ii m K I V S V K v ] [ 1 1 ] [ 1 1 max max 0 (3)
In these equations, [I] is the inhibitor concentration, [S] is the substrate concentration, Km is the Michaelis constant, Vmax is the maximal velocity, and Kii is the overall inhibition constant for the enzyme-substrate-inhibitor complex, and Kis is the overall inhibition constant for the enzyme-inhibitor complex.28
Computational studies
Until date there were no crystal structure reported for
MtIMPDH, which motivate us for protein modeling using one of its close homolog crystal structure with maximum identity as template. The MtIMPDH protein sequence was retrieved from Swiss-Prot protein database (accession id:P9WKI7.1).29 A similarity search was done for the protein sequence against protein data bank using BLAST (Basic Local Alignment Search Tool).30 This search resulted in identification of crystal structure of IMPDH with XMP from Bacillus anthracis str. Ames (PDB ID-3TSD) with 54% identity to MtIMPDH sequence.31 This was the template identified with maximum identity. The homology model for MtIMPDH was generated using Prime v2.3 of Schrödinger 2012.32 Crystal ligand coordinates from template were also incorporated into model during model development. The model generated was subjected to loop refinement and finally the energy was minimized using the OPLS-2005 force field.33 The developed model was inspected for Ramachandran plot using PROCHECK analysis.34 The ProSA analysis was done for the final model so as to study the overall quality of the model.35 The root mean square deviation (rmsd) of model when superimposed over the template was also checked so as to assess the reliability of model. Further, the active site of the model was analyzed based on the sequence alignment and SiteMap v2.6 of Schrödinger 2012.36 More information regarding the homology modeling can be obtained in Supplementary Information.
Molecular docking studies for the synthesized 12 compounds were carried out with the modeled protein using Glide v5.8 of Schrödinger 2012.37 The compounds for docking were sketched using Maestro panel of Schrodinger and their geometry was cleaned and all possible confirmations were generated using LigPrep v2.5 of Schrödinger.37 Finally, the resulted docking poses for the compounds with the protein were analyzed for active site interactions.
Supplementary Information
Supplementary Information (amino acid sequence alignment of MtIMPDH and CpIMPDH, 1H NMR spectra of compounds, 13C NMR spectra of selected compounds, and more details of the computational studies) is available free of charge at http://jbcs.sbq.org.br as PDF file.
Acknowledgements
This work was supported by funds from the National Institute of Science and Technology on Tuberculosis (INCT-TB),
Decit/SCTIE/MS-MCT-CNPq-FNDCT-CAPES (Brazil) to D. S. Santos and L. A. Basso. L. A. Basso and D. S. Santos are Research Career Awardees of the National Research Council of Brazil (CNPq). The fellowships from CNPq (D. C. Rostirolla, K. Pissinate, and L. M. Pinheiro), and from special visiting researcher program are also acknowledged. Finally, P. Yogeeswari acknowledges Department of Biotechnology for their financial assistance.
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Submitted: January 16, 2015
Published online: April 30, 2015
![Table 1. Inhibition of thiazolyl-1H-benzo[d]imidazoles 5a-l on recombinant MtIMPDH activity](https://thumb-eu.123doks.com/thumbv2/123dok_br/18999082.463144/3.892.113.808.104.552/table-inhibition-thiazolyl-benzo-imidazoles-recombinant-mtimpdh-activity.webp)
